The objective of the proposed research is to investigate the significance of cellular bioenergetics to insulin secretion in a bioartificial pancreatic construct composed of transformed beta-cells under environmental conditions that prevail in vivo at the site of implantation. Since transformed beta-cell lines are proposed as alternatives to islets in the development of a bioartificial pancreas, it is imperative that we understand how insulin secretion is affected at these sites. It is our hypothesis that transformed cell lines are more tolerant than mammalian islets to environmental conditions experienced in the peritoneal cavity, a site commonly used for the implantation of these constructs. Experiments proposed in this application will be performed both in vitro and in vivo. Specifically, in vitro experiments will focus on the quantification of glucose metabolism and resultant phosphorylation potential for bioartificial pancreatic constructs composed of betaTC-tet, and INS-1(832/13) cells, as well as porcine islets encapsulated in alginate/poly-L-lysine/alginate (APA) beads as a function of oxygen concentration and pH. Glucose metabolism will be assessed via 13C NMR spectroscopy by measuring the flux through glycolysis, citric acid cycle, and key anaplerotic pathways, while the phosphorylation potential will be quantified via 31P NMR spectroscopy. In vivo experiments will focus on a device that will contain the APA beads so that they are not dispersed throughout the peritoneal cavity and thus NMR experiments performed in vitro can also be performed in vivo. Preliminary data presented in this revised application support our hypothesis and provide the foundation to ensure that proposed experiments are feasible and can be completed successfully. Overall, we believe that the acquired data will contribute significantly to our understanding of the mechanism of insulin secretion, as well as extend our knowledge in the field of tissue engineering.